Beta-glucosidase variants polynucleotides encoding same

ABSTRACT

The present invention relates to beta-glucosidase variants, e.g., beta-glucosidase variants of a parent Family GH3A beta-glucosidase from  Aspergillus fumigatus . The present invention also relates to polynucleotides encoding the beta-glucosidase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the beta-glucosidase variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patent application Ser. No. 13/876,802 filed on Mar. 28, 2013, now U.S. Pat. No. 9,688,975, which is a 35 U.S.C. § 371 national application of PCT/US2011/054185 filed on Sep. 30, 2011, which claims priority or the benefit under 35 U.S.C. § 119 of U.S. Provisional. Application Ser. No. 61/388,997 filed on Oct. 1, 2010, the contents of which are fully incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Cooperative Agreement DE-FC36-08GO18080 awarded by the Department of Energy. The government has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to beta-glucosidase variants, polynucleotides encoding the variants, methods of producing the variants, and methods of using the variants.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked by beta-1,4-bonds. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble beta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and the cleanliness of the ethanol fuel. Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the lignocellulose is converted to fermentable sugars, e.g., glucose, the fermentable sugars are easily fermented by yeast into ethanol.

U.S. Pat. No. 7,244,605 discloses an Aspergillus fumigatus beta-glucosidase and the polynucleotide thereof.

It would be advantageous in the art to improve polypeptides having beta-glucosidase activity for use in the enzymatic degradation of lignocellulosic feedstocks.

The present invention provides variants of a parent beta-glucosidase with improved properties compared to its parent.

SUMMARY OF THE INVENTION

The present invention relates to isolated beta-glucosidase variants, comprising a substitution at one or more (e.g., several) positions corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 2, wherein the variants have beta-glucosidase activity.

The present invention also relates to isolated polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing the variants.

The present invention also relates to methods for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of such a variant having beta-glucosidase activity.

The present invention also relates to methods for producing a fermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition in the presence of such a variant having beta-glucosidase activity;

(b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of such a variant having beta-glucosidase activity.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the genomic DNA sequence and the deduced amino acid sequence of an Aspergillus fumigatus beta-glucosidase gene (SEQ ID NOs: 1 and 2, respectively). The predicted signal peptide is underlined and predicted introns are italicized.

FIG. 2 shows net glucose production of a Trichoderma reesei cellulolytic protein composition with varying concentrations of wild-type Aspergillus fumigatus Family GH3A beta-glucosidase, variant L89M+G91L+F100D+I140V+I186V+S283G+N456E+F512Y, variant L89M+G91L+I140V+I186V, and variant F100D+S283G+N456E+F512Y.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of the present invention, acetylxylan esterase activity is determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Allelic variant: The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase” means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of the present invention, alpha-L-arabinofuranosidase activity is determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40° C. followed by arabinose analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means an alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol. For purposes of the present invention, alpha-glucuronidase activity is determined according to de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase equals the amount of enzyme capable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acid per minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose. For purposes of the present invention, beta-glucosidase activity is determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J. Basic Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0 μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1→4)-xylooligosaccharides, to remove successive D-xylose residues from non-reducing termini. For purposes of the present invention, one unit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolate anion produced per minute at 40° C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20.

cDNA: The term “cDNA” means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the present invention, the Tomme et al. method can be used to determine cellobiohydrolase activity.

Cellulosic material: The term “cellulosic material” means any material containing cellulose. The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. The cellulosic material can be, but is not limited to, agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, and wood (including forestry residue) (see, for example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, New York). It is understood herein that the cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix. In a preferred aspect, the cellulosic material is any biomass material. In another preferred aspect, the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. In another aspect, the cellulosic material is herbaceous material (including energy crops). In another aspect, the cellulosic material is municipal solid waste. In another aspect, the cellulosic material is pulp and paper mill residue. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is wood (including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect, the cellulosic material is bagasse. In another aspect, the cellulosic material is bamboo. In another aspect, the cellulosic material is corn cob. In another aspect, the cellulosic material is corn fiber. In another aspect, the cellulosic material is corn stover. In another aspect, the cellulosic material is miscanthus. In another aspect, the cellulosic material is orange peel. In another aspect, the cellulosic material is rice straw. In another aspect, the cellulosic material is switchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect, the cellulosic material is eucalyptus. In another aspect, the cellulosic material is fir. In another aspect, the cellulosic material is pine. In another aspect, the cellulosic material is poplar. In another aspect, the cellulosic material is spruce. In another aspect, the cellulosic material is willow.

In another aspect, the cellulosic material is algal cellulose. In another aspect, the cellulosic material is bacterial cellulose. In another aspect, the cellulosic material is cotton linter. In another aspect, the cellulosic material is filter paper. In another aspect, the cellulosic material is microcrystalline cellulose. In another aspect, the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. As used herein the term “aquatic biomass” means biomass produced in an aquatic environment by a photosynthesis process. The aquatic biomass can be algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected to pretreatment, using conventional methods known in the art, as described herein. In a preferred aspect, the cellulosic material is pretreated.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or “cellulase” means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic activity include: (1) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481. Total cellulolytic activity is usually measured using insoluble substrates, including Whatman N^(o)1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N^(o)1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in PCS (or other pretreated cellulosic material) for 3-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours, sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acid sequences necessary for expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the variant or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.

Endoglucanase: The term “endoglucanase” means an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to control sequences that provide for its expression.

Family GH3 beta-glucosidase: The term “Family GH3 beta-glucosidase” means a glycoside hydrolase of Family 3 according to Coutinho, P. M. and Henrissat, B., 1999, Carbohydrate-active enzymes: an integrated database approach, in “Recent Advances in Carbohydrate Bioengineering”, H. J. Gilbert, G. Davies, B. Henrissat and B. Svensson eds., The Royal Society of Chemistry, Cambridge, pp. 3-12.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase” or “Family GH61” or “GH61” means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1,4-beta-D-glucanase activity in one family member. The structure and mode of action of these enzymes are non-canonical and they cannot be considered as bona fide glycosidases. However, they are kept in the CAZy classification on the basis of their capacity to enhance the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl) groups from esterified sugar, which is usually arabinose in natural biomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For purposes of the present invention, feruloyl esterase activity is determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals the amount of enzyme capable of releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has beta-glucosidase activity. In one aspect, a fragment contains at least 720 amino acid residues, e.g., at least 760 amino acid residues or at least 800 amino acid residues of SEQ ID NO: 2.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolytic enzyme” or “hemicellulase” means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom, D. and Shoham, Y. Microbial hemicellulases. Current Opinion In Microbiology, 2003, 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates of these enzymes, the hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature, e.g., 50° C., 55° C., or 60° C., and pH, e.g., 5.0 or 5.5.

High stringency conditions: The term “high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

Host cell: The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Increased specific activity: The term “increased specific activity” means a higher enzyme activity per molecule or pmole of a variant compared to the enzyme activity per molecule or pmole of the parent. The increased specific activity of the variant relative to the parent can be assessed, for example, under one or more (e.g., several) conditions of pH, temperature, substrate concentration, and product inhibitor concentration, e.g., glucose.

In one aspect, the condition is pH. For example, the pH can be any pH in the range of 3 to 7, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 (or in between). Any suitable buffer for achieving the desired pH can be used.

In another aspect, the condition is temperature. For example, the temperature can be any temperature in the range of 25° C. to 90° C., e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90° C. (or in between).

In another aspect, the condition is substrate concentration. For example, the substrate concentration can be any substrate concentration in the range of 1 mM to 100 mM, e.g., 1 mM, 10 mM, 25 mM, 50 mM, 75 mM, or 100 mM (or in between). Examples of a substrate include without limitation cellobiose, cellotriose, cellotetrose, beta-gentiobiose, laminaribose, or sophorose.

In another aspect, the condition is product inhibitor concentration. For example, the product inhibitor concentration can be any concentration in the range of 1 mM to 120 mM, e.g., 1 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, or 120 mM (or in between). Examples of a product include without limitation glucose, beta-D-mannose, beta-D-sorbose, or D-glucuronic acid.

In another aspect, a combination of two or more (e.g., several) of the above conditions are used to determine the increased specific activity of the variant relative to the parent, such as any temperature in the range of 25° C. to 90° C., e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90° C. (or in between) at a pH in the range of 3 to 7, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 (or in between).

In one aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 25° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 30° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 35° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 40° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 45° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 50° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 55° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 60° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 65° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 70° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 75° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 80° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 85° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.0 and 90° C.

In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 25° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 30° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 35° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 40° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 45° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 50° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 55° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 60° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 65° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 70° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 75° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 80° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 85° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 4.5 and 90° C.

In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 25° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 30° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 35° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 40° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 45° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 50° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 55° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 60° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 65° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 70° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 75° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 80° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 85° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.0 and 90° C.

In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 25° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 30° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 35° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 40° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 45° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 50° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 55° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 60° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 65° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 70° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 75° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 80° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 85° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 5.5 and 90° C.

In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 25° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 30° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 35° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 40° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 45° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 50° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 55° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 60° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 65° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 70° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 75° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 80° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 85° C. In another aspect, the specific activity of the variant relative to the parent is determined at pH 6.0 and 90° C.

In each of the aspects above, the specific activity of the variant relative to the parent can be determined in the presence of glucose at a concentration of 10 mM. In each of the aspects above, the specific activity of the variant relative to the parent can be determined in the presence of glucose at a concentration of 25 mM. In each of the aspects above, the specific activity of the variant relative to the parent can be determined in the presence of glucose at a concentration of 50 mM. In each of the aspects above, the specific activity of the variant relative to the parent can be determined in the presence of glucose at a concentration of 75 mM. In each of the aspects above, the specific activity of the variant relative to the parent can be determined in the presence of glucose at a concentration of 100 mM. In each of the aspects above, the specific activity of the variant relative to the parent can be determined in the presence of glucose at a concentration of 120 mM.

The increased specific activity of the variant relative to the parent can be determined using any enzyme assay known in the art for beta-glucosidases. Such enzyme assays include without limitation cellobiose or p-nitrophenyl-beta-D-glucopyranoside as a substrate. Alternatively, the increased specific activity of the variant relative to the parent can be determined using any application assay for the variant where the performance of the variant is compared to the parent. For example, the application assay described in Example 5 can be used.

In one aspect, the specific activity of the variant is at least 1.01-fold, e.g., at least 1.05-fold, at least 1.1-fold, at least 1.5-fold, at least 1.8-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, and at least 50-fold higher than the specific activity of the parent.

Isolated: The term “isolated” means a substance in a form or environment which does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 20 to 863 of SEQ ID NO: 2 based on the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) that predicts amino acids 1 to 19 of SEQ ID NO: 2 are a signal peptide. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having beta-glucosidase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 58 to 2580 of SEQ ID NO: 1 based on the SignalP program (Nielsen et al., 1997, supra) that predicts nucleotides 1 to 57 of SEQ ID NO: 1 encode a signal peptide. In another aspect, the mature polypeptide coding sequence is the cDNA sequence contained in nucleotides 58 to 2580 of SEQ ID NO: 1.

Medium stringency conditions: The term “medium stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.

Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

Parent or parent beta-glucosidase: The term “parent” or “parent beta-glucosidase” means a beta-glucosidase to which an alteration is made to produce the variants of the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant thereof, or a fragment thereof.

Polypeptide having cellulolytic enhancing activity: The term “polypeptide having cellulolytic enhancing activity” means a GH61 polypeptide that catalyzes the enhancement of the hydrolysis of a cellulosic material by enzyme having cellulolytic activity. For purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in PCS, wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a polypeptide having cellulolytic enhancing activity for 1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., and pH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST® 1.5L (Novozymes A/S, Bagsværd, Denmark) in the presence of 2-3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014) of cellulase protein loading is used as the source of the cellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” means a cellulosic material derived from corn stover by treatment with heat and dilute sulfuric acid, alkaline pretreatment, or neutral pretreatment.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0, 5.0.0, or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0, 5.0.0, or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5′- and/or 3′-end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having beta-glucosidase activity. In one aspect, a subsequence contains at least 2160 nucleotides, e.g., at least 2280 nucleotides or at least 2400 nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 1.

Variant: The term “variant” means a polypeptide having beta-glucosidase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The variants of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the beta-glucosidase of the mature polypeptide of SEQ ID NO: 2.

Very high stringency conditions: The term “very high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C.

Xylan-containing material: The term “xylan-containing material” means any material comprising a plant cell wall polysaccharide containing a backbone of beta-(1-4)-linked xylose residues. Xylans of terrestrial plants are heteropolymers possessing a beta-(1-4)-D-xylopyranose backbone, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylan may be used. In a preferred aspect, the xylan-containing material is lignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylan degrading activity” or “xylanolytic activity” means a biological activity that hydrolyzes xylan-containing material. The two basic approaches for measuring xylanolytic activity include: (1) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl esterases). Recent progress in assays of xylanolytic enzymes was summarized in several publications including Biely and Puchard, Recent progress in the assays of xylanolytic enzymes, 2006, Journal of the Science of Food and Agriculture 86(11): 1636-1647; Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including, for example, oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans. The most common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan as described in Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3): 257-270. Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activity is defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.

For purposes of the present invention, xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For purposes of the present invention, xylanase activity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activity is defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.

Wild-type polypeptide: The term “wild-type polypeptide” means a beta-glucosidase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated variants, comprising a substitution at one or more (e.g., several) positions corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 2, wherein the variants have beta-glucosidase activity.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another beta-glucosidase. The amino acid sequence of another beta-glucosidase is aligned with the mature polypeptide disclosed in SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in another beta-glucosidase can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using their respective default parameters.

When the other polypeptide has diverged from the mature polypeptide of SEQ ID NO: 2 such that traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide has one or more representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.

For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example, the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single letter or three letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or “G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

Different Alterations.

Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., “Arg170Tyr,Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates the following variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Variants

The present invention provides beta-glucosidase variants comprising a substitution at one or more (e.g., several) positions corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 2, wherein the variants have beta-glucosidase activity.

In an embodiment, the variant has sequence identity of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, to the amino acid sequence of the parent polypeptide having beta-glucosidase activity.

In another embodiment, the variant has at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NO: 2.

In one aspect, the number of substitutions in the variants of the present invention is 1-8, such as 1, 2, 3, 4, 5, 6, 7, or 8 substitutions.

In another aspect, a variant comprises a substitution at one or more (e.g., several) positions corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512. In another aspect, a variant comprises a substitution at two positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512. In another aspect, a variant comprises a substitution at three positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512. In another aspect, a variant comprises a substitution at four positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512. In another aspect, a variant comprises a substitution at five positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512. In another aspect, a variant comprises a substitution at six positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512. In another aspect, a variant comprises a substitution at seven positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512. In another aspect, a variant comprises a substitution at each position corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 89. In another aspect, the amino acid at a position corresponding to position 89 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Met. In another aspect, the variant comprises or consists of the substitution L89M of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 91. In another aspect, the amino acid at a position corresponding to position 91 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu. In another aspect, the variant comprises or consists of the substitution G91L of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 100. In another aspect, the amino acid at a position corresponding to position 100 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Asp. In another aspect, the variant comprises or consists of the substitution F100D of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 140. In another aspect, the amino acid at a position corresponding to position 140 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val. In another aspect, the variant comprises or consists of the substitution I140V of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 186. In another aspect, the amino acid at a position corresponding to position 186 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val. In another aspect, the variant comprises or consists of the substitution I186V of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 283. In another aspect, the amino acid at a position corresponding to position 283 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Gly. In another aspect, the variant comprises or consists of the substitution S283G of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 456. In another aspect, the amino acid at a position corresponding to position 456 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Glu. In another aspect, the variant comprises or consists of the substitution N456E of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 512. In another aspect, the amino acid at a position corresponding to position 512 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Tyr. In another aspect, the variant comprises or consists of the substitution F512Y of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89 and 91, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89 and 100, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89 and 140, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89 and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89 and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89 and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89 and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91 and 100, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91 and 140, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91 and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91 and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91 and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91 and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100 and 140, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100 and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100 and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100 and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100 and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140 and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140 and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140 and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140 and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 186 and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 186 and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 186 and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 283 and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 283 and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 456 and 512, such as those described above.

In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, and 100, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, and 140, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, and 140, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, and 140, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 283, 456, and 512, such as those described above.

In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, and 140, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 186, 283, 456, and 512, such as those described above.

In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, and 186, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 140, 186, 283, 456, and 512, such as those described above.

In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 186, and 283, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 186, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 186, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 140, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 140, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 100, 140, 186, 283, 456, and 512, such as those described above.

In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 186, 283, and 456, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 186, 283, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 186, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 140, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 100, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 91, 140, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 89, 100, 140, 186, 283, 456, and 512, such as those described above. In another aspect, the variant comprises or consists of a substitution at positions corresponding to positions 91, 100, 140, 186, 283, 456, and 512, such as those described above.

In another aspect, the variant comprises or consists of substitutions at positions corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512, such as those described above.

In another aspect, the variant comprises or consists of one or more (e.g., several) substitutions selected from the group consisting of L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y.

In another aspect, the variant comprises or consists of the substitutions L89M+G91L of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions N456E+F512Y of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+I186V of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions I140V+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+I186V+S283G of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+I186V+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+I186V+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+I140V+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+I140V+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions F100D+I140V+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+I186V+S283G+N456E of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+I186V+S283G+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+I186V+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+G91L+I140V+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions L89M+F100D+I140V+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2. In another aspect, the variant comprises or consists of the substitutions G91L+F100D+I140V+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the substitutions L89M+G91L+F100D+I140V+I186V+S283G+N456E+F512Y of the mature polypeptide of SEQ ID NO: 2.

The variants of the present invention may further comprise an alteration, e.g., deletion, insertion, and/or substitution at one or more (e.g., several) other positions.

The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for polypeptide having beta-glucosidase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the polypeptide having beta-glucosidase activity or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

The variants may consist of 720 to 863 amino acids, e.g., 720 to 739, 740 to 759, 760 to 779, 780 to 799, 800 to 819, 820 to 839, and 840 to 863 amino acids.

In an embodiment, the variant has improved specific activity.

Parent Beta-Glucosidases

The parent beta-glucosidase may be may be (a) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under at least low stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii); or (c) a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1. or the cDNA sequence thereof.

In one aspect, the parent has a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have beta-glucosidase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, from the mature polypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ ID NO: 2. In another aspect, the parent comprises or consists of amino acids 20 to 863 of SEQ ID NO: 2.

In another aspect, the parent is a fragment of the mature polypeptide of SEQ ID NO: 2 containing at least 720 amino acid residues, e.g., at least 760 amino acid residues or at least 800 amino acid residues.

In another aspect, the parent is an allelic variant of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the parent is encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1; (iii) the cDNA sequence thereof; (iv) the full-length complement thereof; or (v) a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof. In another aspect, the nucleic acid probe is nucleotides 58 to 2580 of SEQ ID NO: 1 or the cDNA sequence thereof. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1 or the cDNA sequence thereof.

In another aspect, the parent is encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof of at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which encodes a polypeptide having beta-glucosidase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 58 to 2580 of SEQ ID NO: 1.

The parent may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

The parent may be a bacterial beta-glucosidase. For example, the parent may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces beta-glucosidase, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma beta-glucosidase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis beta-glucosidase.

In another aspect, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus beta-glucosidase.

In another aspect, the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans beta-glucosidase.

The parent may be a fungal beta-glucosidase. For example, the parent may be a yeast polypeptide having beta-glucosidase activity such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia beta-glucosidase. For example, the parent may be a filamentous fungal beta-glucosidase such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Dipodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria beta-glucosidase.

In another aspect, the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis beta-glucosidase.

In another aspect, the parent is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chtysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia austra/einsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride beta-glucosidase.

In another aspect, the parent is an Aspergillus fumigatus beta-glucosidase, e.g., the beta-glucosidase of SEQ ID NO: 2 or the mature polypeptide thereof.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding a parent may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a variant having beta-glucosidase activity, comprising: (a) introducing into a parent beta-glucosidase a substitution at one or more (e.g., several) positions corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 2, wherein the variant has beta-glucosidase activity; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g., several) mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encoding a variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which is recognized by a host cell for expression of the polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used.

Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor.

Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a variant of the present invention which increases expression of the variant.

Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′-terminus of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′-terminus of the variant-encoding sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the variant. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a variant. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of the variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the variant would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (e.g., several) convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vector preferably contains one or more (e.g., several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system as described in WO 2010/039889. In one aspect, the dual selectable marker is a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a variant. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide encoding a variant of the present invention operably linked to one or more (e.g., several) control sequences that direct the production of a variant of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the variant and the source of the parent.

The host cell may be any cell useful in the recombinant production of a variant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant, comprising: (a) cultivating a host cell of the present invention under conditions suitable for expression of the variant; and (b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the variant to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that are specific for the variants. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. For example, the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, the whole fermentation broth is recovered.

The variant may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

Compositions

The present invention also relates to compositions comprising a variant of the present invention. Preferably, the compositions are enriched in such a variant. The term “enriched” indicates that the beta-glucosidase activity of the composition has been increased, e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a variant of the present invention as the major enzymatic component, e.g., a mono-component composition. Alternatively, the compositions may comprise multiple enzymatic activities, such as one or more (several) enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art.

The compositions may be a fermentation broth formulation or a cell composition, as described herein. Consequently, the present invention also relates to fermentation broth formulations and cell compositions comprising a variant of the present invention. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.

The term “fermentation broth” as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.

In an embodiment, the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof. In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.

In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.

The fermentation broth formulations or cell compostions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may further comprise one or more enzyme activities such as cellobiohydrolase, endoglucanase, beta-glucosidase, endo-beta-1,3(4)-glucanase, glucohydrolase, xyloglucanase, xylanase, xylosidase, arabinofuranosidase, alpha-glucuronidase, acetyl xylan esterase, mannanase, mannosidase, alpha-galactosidase, mannan acetyl esterase, galactanase, arabinanase, pectate lyase, pectinase lyase, pectate lyase, polygalacturonase, pectin acetyl esterase, pectin methyl esterase, beta-galactosidase, galactanase, arabinanase, alpha-arabinofuranosidase, rhamnogalacturonase, ferrulic acid esterases rhamnogalacturonan lyase, rhamnogalacturonan acetyl esterase, xylogalacturonosidase, xylogalacturonase, rhamnogalacturonan lyase, lignin peroxidases, manganese-dependent peroxidases, hybrid peroxidases, with combined properties of lignin peroxidases and manganese-dependent peroxidases, glucoamylase, amylase, protease, and laccase.

In some embodiments, the cell-killed whole broth or composition includes cellulolytic enzymes including, but not limited to, (i) endoglucanases (EG) or 1,4-D-glucan-4-glucanohydrolases (EC 3.2.1.4), (ii) exoglucanases, including 1,4-D-glucan glucanohydrolases (also known as cellodextnnases) (EC 3.2.1.74) and 1,4-D-glucan cellobiohydrolases (exo-cellobiohydrolases, CBH) (EC 3.2.1.91), and (iii) beta-glucosidase (BG) or beta-glucoside glucohydrolases (EC 3.2.1.21).

The cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)). In some embodiments, the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.

The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.

Examples are given below of preferred uses of the compositions of the present invention. The dosage of the composition and other conditions under which the composition is used may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to the following methods for using the variants, or compositions thereof.

The present invention also relates to methods for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of a variant of the present invention. In one aspect, the methods further comprise recovering the degraded or converted cellulosic material. Soluble products of degradation or conversion of the cellulosic material can be separated from insoluble cellulosic material using any method well known in the art such as, for example, centrifugation, filtration, and/or gravity settling.

The present invention also relates to methods for producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition in the presence of a variant of the present invention; (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a variant of the present invention. In one aspect, the fermenting of the cellulosic material produces a fermentation product. In another aspect, the methods further comprise recovering the fermentation product from the fermentation.

The methods of the present invention can be used to saccharify the cellulosic material to fermentable sugars and to convert the fermentable sugars to many useful fermentation products, e.g., fuel, potable ethanol, and/or platform chemicals (e.g., acids, alcohols, ketones, gases, and the like). The production of a desired fermentation product from the cellulosic material typically involves pretreatment, enzymatic hydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the present invention can be accomplished using processes conventional in the art. Moreover, the methods of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate or simultaneous, include, but are not limited to, separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF); and direct microbial conversion (DMC), also sometimes called consolidated bioprocessing (CBP). SHF uses separate process steps to first enzymatically hydrolyze the cellulosic material to fermentable sugars, e.g., glucose, cellobiose, and pentose monomers, and then ferment the fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of the cellulosic material and the fermentation of sugars to ethanol are combined in one step (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the environment: A strategic perspective on the U.S. Department of Energy's research and development activities for bioethanol, Biotechnol. Prog. 15: 817-827). HHF involves a separate hydrolysis step, and in addition a simultaneous saccharification and hydrolysis step, which can be carried out in the same reactor. The steps in an HHF process can be carried out at different temperatures, i.e., high temperature enzymatic saccharification followed by SSF at a lower temperature that the fermentation strain can tolerate. DMC combines all three processes (enzyme production, hydrolysis, and fermentation) in one or more (e.g., several) steps where the same organism is used to produce the enzymes for conversion of the cellulosic material to fermentable sugars and to convert the fermentable sugars into a final product (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbial cellulose utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein that any method known in the art comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination thereof, can be used in the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, a batch stirred reactor, a continuous flow stirred reactor with ultrafiltration, and/or a continuous plug-flow column reactor (Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensive stirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor types include: fluidized bed, upflow blanket, immobilized, and extruder type reactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatment process known in the art can be used to disrupt plant cell wall components of the cellulosic material (Chandra et al., 2007, Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic materials for efficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility of lignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review, Int. J. of Mol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle size reduction, sieving, pre-soaking, wetting, washing, and/or conditioning prior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment. Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwave, supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gamma irradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/or fermentation. Pretreatment is preferably performed prior to the hydrolysis. Alternatively, the pretreatment can be carried out simultaneously with enzyme hydrolysis to release fermentable sugars, such as glucose, xylose, and/or cellobiose. In most cases the pretreatment step itself results in some conversion of biomass to fermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material is heated to disrupt the plant cell wall components, including lignin, hemicellulose, and cellulose to make the cellulose and other fractions, e.g., hemicellulose, accessible to enzymes. The cellulosic material is passed to or through a reaction vessel where steam is injected to increase the temperature to the required temperature and pressure and is retained therein for the desired reaction time. Steam pretreatment is preferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C., where the optimal temperature range depends on addition of a chemical catalyst. Residence time for the steam pretreatment is preferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10 minutes, where the optimal residence time depends on temperature range and addition of a chemical catalyst. Steam pretreatment allows for relatively high solids loadings, so that the cellulosic material is generally only moist during the pretreatment. The steam pretreatment is often combined with an explosive discharge of the material after the pretreatment, which is known as steam explosion, that is, rapid flashing to atmospheric pressure and turbulent flow of the material to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No. 20020164730). During steam pretreatment, hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes partial hydrolysis of the hemicellulose to monosaccharides and oligosaccharides. Lignin is removed to only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to any chemical pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin. Such a pretreatment can convert crystalline cellulose to amorphous cellulose. Examples of suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation (APR), ionic liquid, and organosolv pretreatments.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) is often added prior to steam pretreatment, which decreases the time and temperature, increases the recovery, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, the cellulosic material is mixed with dilute acid, typically H₂SO₄, and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure. The dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter-current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to, sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium oxide or calcium hydroxide at temperatures of 85-150° C. and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200° C. for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). The pretreatment is performed preferably at 1-40% dry matter, e.g., 2-30% dry matter or 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wet explosion (combination of wet oxidation and steam explosion) can handle dry matter up to 30%. In wet explosion, the oxidizing agent is introduced during pretreatment after a certain residence time. The pretreatment is then ended by flashing to atmospheric pressure (WO 2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic material with liquid or gaseous ammonia at moderate temperatures such as 90-150° C. and high pressure such as 17-20 bar for 5-10 minutes, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018). During AFEX pretreatment cellulose and hemicelluloses remain relatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material by extraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as a catalyst. In organosolv pretreatment, the majority of hemicellulose and lignin is removed.

Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and U.S. Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as a dilute acid treatment, and more preferably as a continuous dilute acid treatment. The acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof. Mild acid treatment is conducted in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in the range from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or 0.1 to 2 wt % acid. The acid is contacted with the cellulosic material and held at a temperature in the range of preferably 140-200° C., e.g., 165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. In preferred aspects, the cellulosic material is present during pretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt % or 30-60 wt %, such as around 40 wt %. The pretreated cellulosic material can be unwashed or washed using any method known in the art, e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanical pretreatment” or “physical pretreatment” refers to any pretreatment that promotes size reduction of particles. For example, such pretreatment can involve various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically) and chemically. Mechanical or physical pretreatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof. In one aspect, high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi. In another aspect, high temperature means temperatures in the range of about 100 to about 300° C., e.g., about 140 to about 200° C. In a preferred aspect, mechanical or physical pretreatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden. The physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjected to physical (mechanical) or chemical pretreatment, or any combination thereof, to promote the separation and/or release of cellulose, hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers to any biological pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the cellulosic material. Biological pretreatment techniques can involve applying lignin-solubilizing microorganisms and/or enzymes (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of cellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Production of ethanol from lignocellulosic materials: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosic material, e.g., pretreated, is hydrolyzed to break down cellulose and/or hemicellulose to fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides. The hydrolysis is performed enzymatically by an enzyme composition in the presence of a variant of the present invention. The components of the compositions can be added simultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions that can be readily determined by one skilled in the art. In one aspect, hydrolysis is performed under conditions suitable for the activity of the enzyme components, i.e., optimal for the enzyme components. The hydrolysis can be carried out as a fed batch or continuous process where the cellulosic material is fed gradually to, for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors or fermentors under controlled pH, temperature, and mixing conditions. Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art. For example, the saccharification can last up to 200 hours, but is typically performed for preferably about 12 to about 120 hours, e.g., about 16 to about 72 hours or about 24 to about 48 hours. The temperature is in the range of preferably about 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about 40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in the range of preferably about 3 to about 8, e.g., about 3.5 to about 7, about 4 to about 6, or about 5.0 to about 5.5. The dry solids content is in the range of preferably about 5 to about 50 wt %, e.g., about 10 to about 40 wt % or about 20 to about 30 wt %.

The enzyme compositions can comprise any protein useful in degrading the cellulosic material.

In one aspect, the enzyme composition comprises or further comprises one or more (e.g., several) proteins selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin. In another aspect, the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. In another aspect, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.

In another aspect, the enzyme composition comprises one or more (e.g., several) cellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (e.g., several) cellulolytic enzymes and one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (e.g., several) enzymes selected from the group of cellulolytic enzymes and hemicellulolytic enzymes. In another aspect, the enzyme composition comprises an endoglucanase. In another aspect, the enzyme composition comprises a cellobiohydrolase. In another aspect, the enzyme composition comprises a beta-glucosidase. In another aspect, the enzyme composition comprises a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises a cellobiohydrolase and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises a beta-glucosidase and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiohydrolase. In another aspect, the enzyme composition comprises an endoglucanase and a beta-glucosidase. In another aspect, the enzyme composition comprises a cellobiohydrolase and a beta-glucosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase, a beta-glucosidase, and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises a cellobiohydrolase, a beta-glucosidase, and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, a beta-glucosidase, and a polypeptide having cellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannan esterase. In another aspect, the enzyme composition comprises an acetylxylan esterase. In another aspect, the enzyme composition comprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect, the enzyme composition comprises an arabinofuranosidase (e.g., alpha-L-arabinofuranosidase). In another aspect, the enzyme composition comprises a coumaric acid esterase. In another aspect, the enzyme composition comprises a feruloyl esterase. In another aspect, the enzyme composition comprises a galactosidase (e.g., alpha-galactosidase and/or beta-galactosidase). In another aspect, the enzyme composition comprises a glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, the enzyme composition comprises a glucuronoyl esterase. In another aspect, the enzyme composition comprises a mannanase. In another aspect, the enzyme composition comprises a mannosidase (e.g., beta-mannosidase). In another aspect, the enzyme composition comprises a xylanase. In a preferred aspect, the xylanase is a Family 10 xylanase. In another aspect, the enzyme composition comprises a xylosidase (e.g., beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. In another aspect, the enzyme composition comprises an expansin. In another aspect, the enzyme composition comprises a laccase. In another aspect, the enzyme composition comprises a ligninolytic enzyme. In a preferred aspect, the ligninolytic enzyme is a manganese peroxidase. In another preferred aspect, the ligninolytic enzyme is a lignin peroxidase. In another preferred aspect, the ligninolytic enzyme is a H₂O₂-producing enzyme. In another aspect, the enzyme composition comprises a pectinase. In another aspect, the enzyme composition comprises a peroxidase. In another aspect, the enzyme composition comprises a protease. In another aspect, the enzyme composition comprises a swollenin.

In the methods of the present invention, the enzyme(s) can be added prior to or during saccharification, saccharification and fermentation, or fermentation.

One or more (e.g., several) components of the enzyme composition may be wild-type proteins, recombinant proteins, or a combination of wild-type proteins and recombinant proteins. For example, one or more (e.g., several) components may be native proteins of a cell, which is used as a host cell to express recombinantly one or more (e.g., several) other components of the enzyme composition. One or more (e.g., several) components of the enzyme composition may be produced as monocomponents, which are then combined to form the enzyme composition. The enzyme composition may be a combination of multicomponent and monocomponent protein preparations.

The enzymes used in the methods of the present invention may be in any form suitable for use, such as, for example, a fermentation broth formulation or a cell composition, a cell lysate with or without cellular debris, a semi-purified or purified enzyme preparation, or a host cell as a source of the enzymes. The enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.

The optimum amounts of the enzymes and a variant of the present invention depend on several factors including, but not limited to, the mixture of cellulolytic and/or hemicellulolytic enzyme components, the cellulosic substrate, the concentration of cellulosic substrate, the pretreatment(s) of the cellulosic substrate, temperature, time, pH, and inclusion of fermenting organism (e.g., yeast for Simultaneous Saccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolytic enzyme protein to cellulosic material is about 0.5 to about 50 mg, preferably at about 0.5 to about 40 mg, more preferably at about 0.5 to about 25 mg, more preferably at about 0.75 to about 20 mg, more preferably at about 0.75 to about 15 mg, even more preferably at about 0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg per g of cellulosic material.

In another aspect, an effective amount of a variant having beta-glucosidase activity to cellulosic material is about 0.01 to about 50.0 mg, preferably about 0.01 to about 40 mg, more preferably about 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg, more preferably about 0.01 to about 10 mg, more preferably about 0.01 to about 5 mg, more preferably at about 0.025 to about 1.5 mg, more preferably at about 0.05 to about 1.25 mg, more preferably at about 0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg, even more preferably at about 0.15 to about 1.25 mg, and most preferably at about 0.25 to about 1.0 mg per g of cellulosic material.

In another aspect, an effective amount of a variant having beta-glucosidase activity to cellulolytic enzyme protein is about 0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g, more preferably at about 0.15 to about 0.75 g, more preferably at about 0.15 to about 0.5 g, more preferably at about 0.1 to about 0.5 g, even more preferably at about 0.1 to about 0.5 g, and most preferably at about 0.05 to about 0.2 g per g of cellulolytic enzyme protein.

The polypeptides having cellulolytic enzyme activity or hemicellulolytic enzyme activity as well as other proteins/polypeptides useful in the degradation of the cellulosic material, e.g., GH61 polypeptides having cellulolytic enhancing activity (collectively hereinafter “polypeptides having enzyme activity”) can be derived or obtained from any suitable origin, including, bacterial, fungal, yeast, plant, or mammalian origin. The term “obtained” also means herein that the enzyme may have been produced recombinantly in a host organism employing methods described herein, wherein the recombinantly produced enzyme is either native or foreign to the host organism or has a modified amino acid sequence, e.g., having one or more (e.g., several) amino acids that are deleted, inserted and/or substituted, i.e., a recombinantly produced enzyme that is a mutant and/or a fragment of a native amino acid sequence or an enzyme produced by nucleic acid shuffling processes known in the art. Encompassed within the meaning of a native enzyme are natural variants and within the meaning of a foreign enzyme are variants obtained recombinantly, such as by site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacillus polypeptide having enzyme activity, or a Gram negative bacterial polypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide having enzyme activity; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide having enzyme activity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaea saccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides having enzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may be a recombinant component, i.e., produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91/17243 and WO 91/17244). The host is preferably a heterologous host (enzyme is foreign to host), but the host may under certain conditions also be a homologous host (enzyme is native to host). Monocomponent cellulolytic proteins may also be prepared by purifying such a protein from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymes comprise a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP (Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Rohm GmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR (Dyadic International, Inc.), or VISCOSTAR® 150L (Dyadic International, Inc.). The cellulase enzymes are added in amounts effective from about 0.001 to about 5.0 wt % of solids, e.g., about 0.025 to about 4.0 wt % of solids or about 0.005 to about 2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods of the present invention, include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (\NO 91/05039; WO 93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); and Thermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the present invention include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichoderma reesei Cel7B endoglucanase I; GENBANK™ accession no. M15665; SEQ ID NO: 4); Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene 63:11-22; Trichoderma reesei CeI5A endoglucanase II; GENBANK™ accession no. M19373; SEQ ID NO: 6); Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accession no. AB003694; SEQ ID NO: 8); Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™ accession no. Z33381; SEQ ID NO: 10); Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884); Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439); Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK™ accession no. L29381); Humicola grisea var. thermoidea endoglucanase (GENBANK™ accession no. AB003107); Melanocarpus albomyces endoglucanase (GENBANK™ accession no. MAL515703); Neurospora crassa endoglucanase (GENBANK™ accession no. XM_324477); Humicola insolens endoglucanase V (SEQ ID NO: 12); Myceliophthora thermophila CBS 117.65 endoglucanase (SEQ ID NO: 14); basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 16); basidiomycete CBS 494.95 endoglucanase (SEQ ID NO: 18); Thielavia terrestris NRRL 8126 CEL6B endoglucanase (SEQ ID NO: 20); Thielavia terrestris NRRL 8126 CEL6C endoglucanase (SEQ ID NO: 22); Thielavia terrestris NRRL 8126 CEL7C endoglucanase (SEQ ID NO: 24); Thielavia terrestris NRRL 8126 CEL7E endoglucanase (SEQ ID NO: 26); Thielavia terrestris NRRL 8126 CEL7F endoglucanase (SEQ ID NO: 28); Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase (SEQ ID NO: 30); and Trichoderma reesei strain No. VTT-D-80133 endoglucanase (SEQ ID NO: 32; GENBANK™ accession no. M15665). The endoglucanases of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32, described above are encoded by the mature polypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31, respectively.

Examples of cellobiohydrolases useful in the present invention include, but are not limited to, Trichoderma reesei cellobiohydrolase I (SEQ ID NO: 34); Trichoderma reesei cellobiohydrolase II (SEQ ID NO: 36); Humicola insolens cellobiohydrolase I (SEQ ID NO: 38); Myceliophthora thermophila cellobiohydrolase II (SEQ ID NO: 40 and SEQ ID NO: 42); Thielavia terrestris cellobiohydrolase II (CEL6A) (SEQ ID NO: 44); Chaetomium thermophilum cellobiohydrolase I (SEQ ID NO: 46); and Chaetomium thermophilum cellobiohydrolase II (SEQ ID NO: 48), Aspergillus fumigatus cellobiohydrolase I (SEQ ID NO: 50), and Aspergillus fumigatus cellobiohydrolase II (SEQ ID NO: 52). The cellobiohydrolases of SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, and SEQ ID NO: 52, described above are encoded by the mature polypeptide coding sequence of SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, and SEQ ID NO: 51, respectively.

Examples of beta-glucosidases useful in the present invention include, but are not limited to, Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2); Aspergillus oryzae beta-glucosidase (SEQ ID NO: 54); Penicillium brasilianum IBT 20888 beta-glucosidase (SEQ ID NO: 56); Aspergillus nigerbeta-glucosidase (SEQ ID NO: 58); and Aspergillus aculeatus beta-glucosidase (SEQ ID NO: 60). The beta-glucosidases of SEQ ID NO: 2, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 58, and SEQ ID NO: 60, described above are encoded by the mature polypeptide coding sequence of SEQ ID NO: 2, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 59, respectively.

Examples of other beta-glucosidases useful in the present invention include a Aspergillus oryzae beta-glucosidase variant fusion protein of SEQ ID NO: 62 or the Aspergillus oryzae beta-glucosidase fusion protein of SEQ ID NO: 64. The beta-glucosidase fusion proteins of SEQ ID NO: 122 and SEQ ID NO: 124 are encoded by SEQ ID NO: 61 and SEQ ID NO: 63, respectively.

The Aspergillus oryzae beta-glucosidase can be obtained according to WO 2002/095014. The Aspergillus fumigatus beta-glucosidase can be obtained according to WO 2005/047499. The Penicillium brasilianum beta-glucosidase can be obtained according to WO 2007/019442. The Aspergillus niger beta-glucosidase can be obtained according to Dan et al., 2000, J. Biol. Chem. 275: 4973-4980. The Aspergillus aculeatus beta-glucosidase can be obtained according to Kawaguchi et al., 1996, Gene 173: 287-288.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidases are disclosed in numerous Glycosyl Hydrolase families using the classification according to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696.

Other cellulolytic enzymes that may be used in the present invention are described in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,648,263, and U.S. Pat. No. 5,686,593.

In the methods of the present invention, any GH61 polypeptide having cellulolytic enhancing activity can be used.

In a first aspect, the GH61 polypeptide having cellulolytic enhancing activity comprises the following motifs:

(SEQ ID NO: 145 or SEQ ID NO: 146) [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and  [FW]-[TF]-K-[AIV], wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 contiguous positions, and X(4) is any amino acid at 4 contiguous positions.

The isolated polypeptide comprising the above-noted motifs may further comprise:

(SEQ ID NO: 147 or SEQ ID NO: 148) H-X(1,2)-G-P-X(3)-[YW]-[AILMV], (SEQ ID NO: 149) [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],  or (SEQ ID NO: 150 or SEQ ID NO: 151) H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and  (SEQ ID NO: 152) [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2 contiguous positions, X(3) is any amino acid at 3 contiguous positions, and X(2) is any amino acid at 2 contiguous positions. In the above motifs, the accepted IUPAC single letter amino acid abbreviation is employed.

In a preferred embodiment, the isolated GH61 polypeptide having cellulolytic enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YVV]-[AILMV] (SEQ ID NO: 153 or SEQ ID NO: 154). In another preferred embodiment, the isolated GH61 polypeptide having cellulolytic enhancing activity further comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 155). In another preferred embodiment, the isolated GH61 polypeptide having cellulolytic enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YVV]-[AILMV] (SEQ ID NO: 156 or SEQ ID NO: 157) and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 158).

In a second aspect, isolated polypeptides having cellulolytic enhancing activity, comprise the following motif:

(SEQ ID NO: 159 or SEQ ID NO: 160) [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A- [HNQ],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 contiguous positions, and X(3) is any amino acid at 3 contiguous positions. In the above motif, the accepted IUPAC single letter amino acid abbreviation is employed.

In a third aspect, the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence that has a degree of sequence identity to the mature polypeptide of SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, or SEQ ID NO: 128 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

In a fourth aspect, the GH61 polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide that hybridizes under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, or SEQ ID NO: 127, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, or SEQ ID NO: 79, or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, or SEQ ID NO: 127, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, or SEQ ID NO: 127 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has cellulolytic enhancing activity.

In a fifth aspect, the GH61 polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide comprising or consisting of a nucleotide sequence that has a degree of sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, or SEQ ID NO: 127 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

In a sixth aspect, the GH61 polypeptide having cellulolytic enhancing activity is an artificial variant comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, or SEQ ID NO: 128; or a homologous sequence thereof.

Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for cellulolytic enhancing activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to the parent polypeptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, or SEQ ID NO: 128 is not more than 4, e.g., 1, 2, 3, or 4.

In one aspect, the GH61 polypeptide having cellulolytic enhancing activity is used in the presence of a soluble activating divalent metal cation according to WO 2008/151043, e.g., manganese sulfate.

In another aspect, the GH61 polypeptide having cellulolytic enhancing activity is used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pretreated cellulosic material such as pretreated corn stover (PCS).

The dioxy compound may include any suitable compound containing two or more oxygen atoms. In some aspects, the dioxy compounds contain a substituted aryl moiety as described herein. The dioxy compounds may comprise one or more (e.g., several) hydroxyl and/or hydroxyl derivatives, but also include substituted aryl moieties lacking hydroxyl and hydroxyl derivatives. Non-limiting examples of the dioxy compounds include pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid; methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone; 2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol; (croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone; cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione; dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate; 4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or a salt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ring system as described herein. The compounds may comprise one or more (e.g., several) additional rings, and are not limited to a specific number of rings unless otherwise stated. In one aspect, the bicyclic compound is a flavonoid. In another aspect, the bicyclic compound is an optionally subsituted isoflavonoid. In another aspect, the bicyclic compound is an optionally substituted flavylium ion, such as an optionally substituted anthocyanidin or optionally substituted anthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as an optionally substituted aromatic or non-aromatic ring comprising a heteroatom, as described herein. In one aspect, the heterocyclic is a compound comprising an optionally substituted heterocycloalkyl moiety or an optionally substituted heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted 5-membered heterocycloalkyl or an optionally substituted 5-membered heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl or optionally substituted heteroaryl moiety is an optionally substituted moiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl, piperidinyl, and oxepinyl. In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted furanyl. Non-limiting examples of the heterocyclic compounds include (1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one; 4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone; [1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone; ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconic acid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran; 5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate thereof.

The nitrogen-containing compound may be any suitable compound with one or more nitrogen atoms. In one aspect, the nitrogen-containing compound comprises an amine, imine, hydroxylamine, or nitroxide moiety. Non-limiting examples of thenitrogen-containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol; 1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; and maleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinone moiety as described herein. Non-limiting examples of the quinone compounds include 1,4-benzoquinone; 1,4-naphthoquinone; 2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Qo; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; 1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or adrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinoline quinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprising one or more sulfur atoms. In one aspect, the sulfur-containing comprises a moiety selected from thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limiting examples of the sulfur-containing compounds include ethanethiol; 2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid; benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione; cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁵ to about 1, about 10⁻⁵ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³ to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, an effective amount of such a compound described above is about 0.1 μM to about 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM to about 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, or a combination thereof, arising from treatment of a lignocellulose and/or hemicellulose material in a slurry, or monosaccharides thereof, e.g., xylose, arabinose, mannose, etc., under conditions as described herein, and the soluble contents thereof. A liquor for cellulolytic enhancement of a GH61 polypeptide can be produced by treating a lignocellulose or hemicellulose material (or feedstock) by applying heat and/or pressure, optionally in the presence of a catalyst, e.g., acid, optionally in the presence of an organic solvent, and optionally in combination with physical disruption of the material, and then separating the solution from the residual solids. Such conditions determine the degree of cellulolytic enhancement obtainable through the combination of liquor and a GH61 polypeptide during hydrolysis of a cellulosic substrate by a cellulase preparation. The liquor can be separated from the treated material using a method standard in the art, such as filtration, sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about 10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about 1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ to about 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² g per g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymes comprise a commercial hemicellulolytic enzyme preparation. Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYME™ (Novozymes A/S), CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (Novozymes A/S), PULPZYME® HC (Novozymes A/S), MULTIFECTO Xylanase (Genencor), ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the methods of the present invention include, but are not limited to, xylanases from Aspergillus (GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO 2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp. (WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), and Trichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the methods of the present invention include, but are not limited to, Trichoderma reesei beta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromyces emersonii (SwissProt accession number Q8X212), and Neurospora crassa (SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the present invention include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprot accession number Q2GWX4), Chaetomium gracile (GeneSeqP accession number AAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880), Neurospora crassa (UniProt accession number q7s259), Phaeosphaeria nodorum (Uniprot accession number Q0UHJ1), and Thielavia terrestris NRRL 8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in the methods of the present invention include, but are not limited to, feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122), Neosartorya fischeri (UniProt Accession number A1D9T4), Neurospora crassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases useful in the methods of the present invention include, but are not limited to, arabinofuranosidases from Aspergillus niger (GeneSeqP accession number AAR94170), Humicola insolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases useful in the methods of the present invention include, but are not limited to, alpha-glucuronidases from Aspergillus clavatus (UniProt accession number alcc12), Aspergillus fumigatus (SwissProt accession number Q4WW45), Aspergillus niger (Uniprot accession number Q96WX9), Aspergillus terreus (SwissProt accession number Q0CJP9), Humicola insolens (WO 2010/014706), Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii (UniProt accession number Q8X211), and Trichoderma reesei (Uniprot accession number Q99024).

The polypeptides having enzyme activity used in the methods of the present invention may be produced by fermentation of the above-noted microbial strains on a nutrient medium containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., Bennett, J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press, C A, 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). Temperature ranges and other conditions suitable for growth and enzyme production are known in the art (see, e.g., Bailey, J. E., and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting in the expression or isolation of an enzyme or protein. Fermentation may, therefore, be understood as comprising shake flask cultivation, or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzyme to be expressed or isolated. The resulting enzymes produced by the methods described above may be recovered from the fermentation medium and purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic material can be fermented by one or more (e.g., several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a desired fermentation product. “Fermentation” or “fermentation process” refers to any fermentation process or any process comprising a fermentation step. Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. The fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast. Hydrolysis (saccharification) and fermentation can be separate or simultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in the fermentation step in practicing the present invention. The material is generally selected based on the desired fermentation product, i.e., the substance to be obtained from the fermentation, and the process employed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a medium before the fermenting microorganism(s) is(are) added, such as, a medium resulting from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art. Suitable fermenting microorganisms are able to ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugars include bacterial and fungal organisms, such as yeast. Preferred yeast includes strains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars in their native state include bacterial and fungal organisms, such as some yeast. Preferred xylose fermenting yeast include strains of Candida, preferably C. sheatae or C. sonorensis; and strains of Pichia, preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentose fermenting yeast include strains of Pachysolen, preferably P. tannophilus. Organisms not capable of fermenting pentose sugars, such as xylose and arabinose, may be genetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexose and pentose to ethanol include, for example, Bacillus coagulans, Clostridium acetobutylicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonas mobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacillus coagulans; Candida, such as C. sonorensis, C. methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis, and C. scehatae; Clostridium, such as C. acetobutylicum, C. thermocellum, and C. phytofermentans; E. coli, especially E. coli strains that have been genetically modified to improve the yield of ethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala; Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K. lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such as S. pombe; Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferred aspect, the yeast is Bretannomyces clausenii. In another preferred aspect, the yeast is a Candida. In another more preferred aspect, the yeast is Candida sonorensis. In another more preferred aspect, the yeast is Candida boidinii. In another more preferred aspect, the yeast is Candida blankii. In another more preferred aspect, the yeast is Candida brassicae. In another more preferred aspect, the yeast is Candida diddensii. In another more preferred aspect, the yeast is Candida entomophiliia. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida scehatae. In another more preferred aspect, the yeast is Candida utilis. In another preferred aspect, the yeast is a Clavispora. In another more preferred aspect, the yeast is Clavispora lusitaniae. In another more preferred aspect, the yeast is Clavispora opuntiae. In another preferred aspect, the yeast is a Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another more preferred aspect, the yeast is Kluyveromyces marxianus. In another more preferred aspect, the yeast is Kluyveromyces thermotolerans. In another preferred aspect, the yeast is a Pachysolen. In another more preferred aspect, the yeast is Pachysolen tannophilus. In another preferred aspect, the yeast is a Pichia. In another more preferred aspect, the yeast is a Pichia stipitis. In another preferred aspect, the yeast is a Saccharomyces spp. In a more preferred aspect, the yeast is Saccharomyces cerevisiae. In another more preferred aspect, the yeast is Saccharomyces distaticus. In another more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferred aspect, the bacterium is Bacillus coagulans. In another preferred aspect, the bacterium is a Clostridium. In another more preferred aspect, the bacterium is Clostridium acetobutylicum. In another more preferred aspect, the bacterium is Clostridium phytofermentans. In another more preferred aspect, the bacterium is Clostridium thermocellum. In another more preferred aspect, the bacterium is Geobacillus sp. In another more preferred aspect, the bacterium is a Thermoanaerobacter. In another more preferred aspect, the bacterium is Thermoanaerobacter saccharolyticum. In another preferred aspect, the bacterium is a Zymomonas. In another more preferred aspect, the bacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include, e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation, GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™ (Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™ (Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast (Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganisms has led to the construction of organisms capable of converting hexoses and pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TALI genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase, Appl. Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle, FEMS Yeast Research 4: 655-664; Beall et al., 1991, Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli, Biotech. Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganism is Candida sonorensis. In another preferred aspect, the genetically modified fermenting microorganism is Escherichia coli. In another preferred aspect, the genetically modified fermenting microorganism is Klebsiella oxytoca. In another preferred aspect, the genetically modified fermenting microorganism is Kluyveromyces marxianus. In another preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can also be used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degraded cellulosic material or hydrolysate and the fermentation is performed for about 8 to about 96 hours, e.g., about 24 to about 60 hours. The temperature is typically between about 26° C. to about 60° C., e.g., about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.

In one aspect, the yeast and/or another microorganism are applied to the degraded cellulosic material and the fermentation is performed for about 12 to about 96 hours, such as typically 24-60 hours. In another aspect, the temperature is preferably between about 20° C. to about 60° C., e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., or about 32° C. to about 50° C., and the pH is generally from about pH 3 to about pH 7, e.g., about pH 4 to about pH 7. However, some fermenting organisms, e.g., bacteria, have higher fermentation temperature optima. Yeast or another microorganism is preferably applied in amounts of approximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰, especially approximately 2×10⁸ viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurry is distilled to extract the ethanol. The ethanol obtained according to the methods of the invention can be used as, e.g., fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of the methods described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield. A “fermentation stimulator” refers to stimulators for growth of the fermenting microorganisms, in particular, yeast. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E. See, for example, Alfenore et al., Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), which is hereby incorporated by reference. Examples of minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from the fermentation. The fermentation product can be, without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); an organic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); and polyketide. The fermentation product can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It will be understood that the term “alcohol” encompasses a substance that contains one or more hydroxyl moieties. In a more preferred aspect, the alcohol is n-butanol. In another more preferred aspect, the alcohol is isobutanol. In another more preferred aspect, the alcohol is ethanol. In another more preferred aspect, the alcohol is methanol. In another more preferred aspect, the alcohol is arabinitol. In another more preferred aspect, the alcohol is butanediol. In another more preferred aspect, the alcohol is ethylene glycol. In another more preferred aspect, the alcohol is glycerin. In another more preferred aspect, the alcohol is glycerol. In another more preferred aspect, the alcohol is 1,3-propanediol. In another more preferred aspect, the alcohol is sorbitol. In another more preferred aspect, the alcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes for fermentative production of xylitol—a sugar substitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping, World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. The alkane can be an unbranched or a branched alkane. In another more preferred aspect, the alkane is pentane. In another more preferred aspect, the alkane is hexane. In another more preferred aspect, the alkane is heptane. In another more preferred aspect, the alkane is octane. In another more preferred aspect, the alkane is nonane. In another more preferred aspect, the alkane is decane. In another more preferred aspect, the alkane is undecane. In another more preferred aspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane. In another more preferred aspect, the cycloalkane is cyclopentane. In another more preferred aspect, the cycloalkane is cyclohexane. In another more preferred aspect, the cycloalkane is cycloheptane. In another more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. The alkene can be an unbranched or a branched alkene. In another more preferred aspect, the alkene is pentene. In another more preferred aspect, the alkene is hexene. In another more preferred aspect, the alkene is heptene. In another more preferred aspect, the alkene is octene.

In another preferred aspect, the fermentation product is an amino acid. In another more preferred aspect, the organic acid is aspartic acid. In another more preferred aspect, the amino acid is glutamic acid. In another more preferred aspect, the amino acid is glycine. In another more preferred aspect, the amino acid is lysine. In another more preferred aspect, the amino acid is serine. In another more preferred aspect, the amino acid is threonine. See, for example, Richard, A., and Margaritis, A., 2004, Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. In another more preferred aspect, the gas is methane. In another more preferred aspect, the gas is H₂. In another more preferred aspect, the gas is CO₂. In another more preferred aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. It will be understood that the term “ketone” encompasses a substance that contains one or more ketone moieties. In another more preferred aspect, the ketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.

In another preferred aspect, the fermentation product is an organic acid. In another more preferred aspect, the organic acid is acetic acid. In another more preferred aspect, the organic acid is acetonic acid. In another more preferred aspect, the organic acid is adipic acid. In another more preferred aspect, the organic acid is ascorbic acid. In another more preferred aspect, the organic acid is citric acid. In another more preferred aspect, the organic acid is 2,5-diketo-D-gluconic acid. In another more preferred aspect, the organic acid is formic acid. In another more preferred aspect, the organic acid is fumaric acid. In another more preferred aspect, the organic acid is glucaric acid. In another more preferred aspect, the organic acid is gluconic acid. In another more preferred aspect, the organic acid is glucuronic acid. In another more preferred aspect, the organic acid is glutaric acid. In another preferred aspect, the organic acid is 3-hydroxypropionic acid. In another more preferred aspect, the organic acid is itaconic acid. In another more preferred aspect, the organic acid is lactic acid. In another more preferred aspect, the organic acid is malic acid. In another more preferred aspect, the organic acid is malonic acid. In another more preferred aspect, the organic acid is oxalic acid. In another more preferred aspect, the organic acid is propionic acid. In another more preferred aspect, the organic acid is succinic acid. In another more preferred aspect, the organic acid is xylonic acid. See, for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol is separated from the fermented cellulosic material and purified by conventional methods of distillation. Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

Plants

The present invention also relates to plants, e.g., a transgenic plant, plant part, or plant cell, comprising a polynucleotide of the present invention so as to express and produce the variant in recoverable quantities. The variant may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the variant may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilization of the invention are also considered plant parts, e.g., embryos, endosperms, aleurone and seed coats.

Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant may be constructed in accordance with methods known in the art. In short, the plant or plant cell is constructed by incorporating one or more (e.g., several) expression constructs encoding a variant into the plant host genome or chloroplast genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct that comprises a polynucleotide encoding a variant operably linked with appropriate regulatory sequences required for expression of the polynucleotide in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying plant cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, on the basis of when, where, and how the variant is desired to be expressed. For instance, the expression of the gene encoding a variant may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiol. 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, and the rice actin 1 promoter may be used (Franck et al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (VVu et al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higher expression of a variant in the plant. For instance, the promoter enhancer element may be an intron that is placed between the promoter and the polynucleotide encoding a variant. For instance, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can also be used for transforming monocots, although other transformation methods are often used for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformation methods for use in accordance with the present disclosure include those described in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are herein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype with a construct prepared according to the present invention, transgenic plants may be made by crossing a plant having the construct to a second plant lacking the construct. For example, a construct encoding a variant can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the present invention encompasses not only a plant directly regenerated from cells which have been transformed in accordance with the present invention, but also the progeny of such plants. As used herein, progeny may refer to the offspring of any generation of a parent plant prepared in accordance with the present invention. Such progeny may include a DNA construct prepared in accordance with the present invention, or a portion of a DNA construct prepared in accordance with the present invention. Crossing results in the introduction of a transgene into a plant line by cross pollinating a starting line with a donor plant line. Non-limiting examples of such steps are further articulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. For example, plants include plants referred to as a backcross converted genotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one or more transgenes of the invention from one genetic background into another. Marker assisted selection offers advantages relative to conventional breeding in that it can be used to avoid errors caused by phenotypic variations. Further, genetic markers may provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait which otherwise has a non-agronomically desirable genetic background is crossed to an elite parent, genetic markers may be used to select progeny which not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits into a particular genetic background is minimized.

The present invention also relates to methods of producing a variant of the present invention comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the variant under conditions conducive for production of the variant; and (b) recovering the variant.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Strains

Plasmid pEJG97 (WO 2005/074647 was used as the source of DNA encoding the Aspergillus fumigatus Family 3A beta-glucosidase. Aspergillus oryzae JaL250 strain (WO 99/61651) was used for expression of the A. fumigatus Family 3A beta-glucosidase and variants thereof.

Media

PDA plates were composed of 39 grams of potato dextrose agar and deionized water to 1 liter.

MDU2BP medium was composed of 45 g of maltose, 1 g of MgSO₄.7H₂O, 1 g of NaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeast extract, 2 g of urea, 0.5 ml of AMG trace metals solution, and deionized water to 1 liter, pH to 5.0.

AMG trace metals solution was composed of 14.3 g of ZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 3 g of citric acid, and deionized water to 1 liter.

Example 1: Preparation of Aspergillus fumigatus GH3A Beta-Glucosidase

The A. fumigatus GH3A beta-glucosidase (SEQ ID NO: 1 [DNA sequence] and SEQ ID NO: 2 [deduced amino acid sequence]) as shown in FIGS. 1A and 1B was prepared according to U.S. Pat. No. 7,244,605. Protein concentration was determined using a Microplate BOA™ Protein Assay Kit (Thermo Fischer Scientific, Waltham, Mass., USA) in which bovine serum albumin was used as a protein standard.

Example 2: Construction of a Aspergillus fumigatus Family GH3A Beta-Glucosidase Variant

Variants of the Aspergillus fumigatus Family 3A beta-glucosidase were constructed by performing site-directed mutagenesis on pEJG97 using a QUIKCHANGE® Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA).

A summary of the oligos used for the site-directed mutagenesis and the variants obtained are shown in Table 1.

The resulting mutant plasmid DNA was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA). The variant plasmid construct was sequenced using an Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA) to verify the changes.

TABLE 1 Amino Cloning acid Plasmid changes Primer name Sequences Name F100D AfBGmutF100DF ccctttgggtatccgtGACtgtgagctatacccgcg pDFng128-6 (SEQ ID NO: 129) S283G AfBGmutS283GF cgtcatgagtgactggGGCgctcaccacagcggtg (SEQ ID NO: 130) N456E AfBGmutN456EF gggtagtggtactgccGAGttcccttaccttgtcac (SEQ ID NO: 131) F512Y AfBGmutF512YF gccgactctggagagggtTACatcagtgtcgacggcaac (SEQ ID NO: 132)

Example 3: Expression of the Aspergillus Fumigatus Family GH3A Beta-Glucosidase Variant F100D+S283G+N456E+F512Y in Aspergillus oryzae JaL250

Aspergillus oryzae JaL250 protoplasts were prepared according to the method of Christensen et al., 1988, Bio/Technology 6: 1419-1422, and transformed with 5 μg of expression vector pDFng128-6 or pEJG97 as a control. The transformation of Aspergillus oryzae JaL250 with pDFng128-6 or pEJG97 yielded multiple transformants. Five transformants for each transformation were isolated to individual PDA plates.

Confluent PDA plates of all transformants were washed with 8 ml of 0.01% TWEEN® 20 and inoculated separately into 1 ml of MDU2BP medium in sterile 24 well tissue culture plates and incubated at 34° C. Five days after incubation, a 10 μl sample from harvested broth of each culture was analyzed using 8-16% Tris-glycine SDS-PAGE gels (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's instructions. SDS-PAGE profiles of the cultures showed that several transformants had a new major band of approximately 100 kDa.

A confluent PDA plate of one transformant for each transformation was washed with 8 ml of 0.01% TWEEN® 20 and inoculated into 500 ml glass shake flasks containing 100 ml of MDU2BP medium and incubated at 34° C., 200 rpm to generate broth for characterization of the enzyme. The flasks were harvested on day 5 and filtered using a 0.22 μm GP Express plus Membrane (Millipore, Bedford, Mass., USA).

Example 4: Purification of the Wild-Type Aspergillus fumigatus Family GH3A Beta Glucosidase and Variant F100D+S283G+N456E+F512Y

Broth supernatant containing either wild-type A. fumigatus GH3A beta-glucosidase, obtained according to Example 1, or A. fumigatus GH3A beta-glucosidase variant F100D+S283G+N456E+F512Y, obtained according to Example 2, was concentrated and desalted using ECONOPAC® 10 DG columns (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) equilibrated with 20 mM Tris-HCl pH 8.0. A 3 ml volume of broth supernatant was loaded onto the column and then eluted with a 4 ml volume of 20 mM Tris-HCl pH 8.0. The desalted sample was purified using a 20 ml MONO Q™ column (HR 16/10, GE Healthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris-HCl pH 8.0. A 16 ml volume of the desalted material was loaded onto the MONO Q™ column and then washed with 5 column volumes of 20 mM Tris-HCl pH 8.0. The wild-type A. fumigatus GH3A beta-glucosidase or A. fumigatus GH3A beta-glucosidase variant was eluted using a 20 column volume gradient from 0.16 to 0.40 M NaCl in 20 mM Tris-HCl pH 8.0. Fractions of 10 ml were collected, and fractions 11 through 21 were pooled for the variant and fractions 10 through 20 were pooled for the wild-type enzyme. Purified samples were submitted to SDS-PAGE analysis. SDS-PAGE on a 8-16% stain free Tris-HCl gel (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) with imaging using a CRITERION® Stain Free Imager (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) showed the samples to be greater than 95% pure.

Example 5: Pretreatment of Corn Stover

Corn stover was pretreated at the U.S. Department of Energy National Renewable Energy Laboratory (NREL) using 1.4% (w/v) sulfuric acid for 2 minutes at 190° C. The water-insoluble solids in the pretreated corn stover (PCS) contained 57.5% cellulose, 4.6% hemicelluloses, and 28.4% lignin. Cellulose and hemicellulose were determined by a two-stage sulfuric acid hydrolysis with subsequent analysis of sugars by high performance liquid chromatography using NREL Standard Analytical Procedure #002. Lignin was determined gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid using NREL Standard Analytical Procedure #003.

The pretreated corn stover was washed repeatedly using reverse osmosis water followed by decanting off the supernatant fraction, until the measured pH was greater than 4.0. The washed pretreated corn stover (initial dry weight 32.35% TS) was subsequently milled prior to use in a Cosmos ICMG 40 wet multi-utility grinder (EssEmm Corporation, Tamil Nadu, India). The dry weight of the milled, water-washed pretreated corn stover was 7.4% TS.

Example 6: Determination of the Specific Activity of the Wild-Type Aspergillus fumigatus GH3A Beta-Glucosidase and Variant F100D+S283G+N456E+F512Y

Purified wild-type Aspergillus fumigatus GH3A beta-glucosidase and variant F100D+S283G+N456E+F512Y, purified according to Examples 1 and 2, respectively, were evaluated for their ability to enhance the hydrolysis of PCS by a Trichoderma reesei cellulolytic protein preparation composed of CELLUCLAST® 1.5L (Novozymes A/S, Bagsvaerd, Denmark) supplemented with 10% addition of Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (obtained as described in WO 2005/074656) and 2% addition of SHEARZYME® 2X (Novozymes A/S, Bagsvaerd, Denmark), hereinafter designated “Trichoderma reesei cellulolytic protein composition”.

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates (Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml. The hydrolysis was performed with 50 mg of washed, ground, sieved PCS (Example 5) per ml of 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate, 40 g of glucose per ml, and a fixed protein loading of 2.24 mg of the T. reesei cellulolytic protein composition per gram of cellulose and between 0 to 10% addition (by protein) to the base CELLUCLAST 1.5L enzyme loading with the purified Aspergillus fumigatus Family 3A beta-glucosidase (2.24 mg of the T. reesei cellulolytic protein composition per g of cellulose and between 0 and 0.2 mg of Aspergillus fumigatus Family 3A beta-glucosidase or variant thereof per g of cellulose). Hydrolysis assays were performed in triplicate for 72 hours at 50° C. Following hydrolysis, samples were filtered with a 0.45 μm MULTISCREEN® 96-well filter plate (Millipore, Bedford, Mass., USA) and filtrates analyzed for sugar content as described below. When not used immediately, filtered sugary aliquots were frozen at −20° C.

Sugar concentrations of samples diluted in 0.005 M H₂SO₄ were measured after elution by 0.005 M H₂SO₄ with 0.05% w/w benzoic acid at a flow rate of 0.6 ml per minute from a 4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) at 65° C. with quantitation by integration of glucose and cellobiose signals from refractive index detection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, Santa Clara, Calif., USA) calibrated by pure sugar samples. The resultant equivalents were used to calculate the percentage of cellulose conversion for each reaction.

Glucose released was calculated by subtracting glucose levels measured in the PCS alone from those measured in PCS with enzyme mixture.

The results shown in FIG. 2 demonstrated that the A. fumigatus beta-glucosidase variant F100D+S283G+N456E+F512Y has a higher specific activity than the wild-type beta-glucosidase (releases more glucose at a given enzyme concentration than the wild-type enzyme at the same concentration under the specified conditions).

Example 7: Construction of a Aspergillus fumigatus Family GH3A Beta-Glucosidase Gene Variants

Variants of the Aspergillus fumigatus Family 3A beta-glucosidase were constructed by performing site-directed mutagenesis on pEJG97 (for pMaWo87i1) or pDFng128-6 (for pMaWo86i1) or pMaWo87i1 (for pMaWo87i2) or pMaWo86i1 (for pMaWo86i2) or pMaWo86i2 (for pMaWo86) or pMaWo87i2 (for pMaWo87) using a QUIKCHANGE® Site-Directed Mutagenesis Kit.

A summary of the oligos used for the site-directed mutagenesis and the variants obtained are shown in Table 2.

The resulting mutant plasmid DNAs were prepared using a BIOROBOT® 9600. The variant plasmid constructs were sequenced using an Applied Biosystems 3130xl Genetic Analyzer to verify the changes.

TABLE 2 Amino acid Primer Cloning changes name Sequences Plasmid Name I140V MaWo220 cgacaagggcgtggacgttttgctggggcctgc pMaWo87i1 (SEQ ID NO: 133) MaWo221 gcaggccccagcaaaacgtccacgcccttgtcg (SEQ ID NO: 134) I140V + F100D + MaWo220 cgacaagggcgtggacgttttgctggggcctgc pMaWo86i1 S283G + N456E + (SEQ ID NO: 135) F512Y MaWo221 gcaggccccagcaaaacgtccacgcccttgtcg (SEQ ID NO: 136) I186V + I140V MaWo222 ccaagacgcgggtgtggttgctactgccaagc pMaWo87i2 (SEQ ID NO: 137) MaWo223 gcttggcagtagcaaccacacccgcgtcttgg (SEQ ID NO: 138) L89M + G91L + MaWo218 ggtatcaactggggtatgtgtctccaggattcccctttggg pMaWo86i2 I140V + F100D + tatccg (SEQ ID NO: 139) S283G + N456E + MaWo219 cggatacccaaaggggaatcctggagacacataccccagtt F512Y gatacc (SEQ ID NO: 140) L89M + G91L + MaWo218 ggtatcaactggggtatgtgtctccaggattcccctttggg pMaWo87 I186V + I140V tatccg (SEQ ID NO: 141) MaWo219 cggatacccaaaggggaatcctggagacacataccccagtt gatacc (SEQ ID NO: 142) I186V + L89M + MaWo222 ccaagacgcgggtgtggttgctactgccaagc pMaWo86 G91L + I140V + (SEQ ID NO: 143) F100D + S283G + MaWo223 gcttggcagtagcaaccacacccgcgtcttgg N456E + F512Y (SEQ ID NO: 144)

Example 8: Expression of the Aspergillus fumigatus Family GH3A Beta-Glucosidase Variant L89M+G91L+F100D+I140V+I186V+S283G+N456E+F512Y, and Variant L89M+G91L+I140V+I186V in Aspergillus oryzae JaL250

Aspergillus oryzae JaL250 protoplasts were prepared according to the method of Christensen et al., 1988, supra, and transformed with 5 μg of expression vector pMaWo86 or pMaWo87 or pEJG97 (as a control) or pDFng128-6 (as a control). The transformation of Aspergillus oryzae JaL250 with pDFng128-6 or pEJG97 yielded multiple transformants. Five transformants for each transformation were isolated to individual PDA plates. The transformation of Aspergillus oryzae JaL250 with pMaWo86 yielded four transformants, which were also isolated to individual PDA plates. The transformation of Aspergillus oryzae JaL250 with pMaWo87 yielded two transformants, which were also isolated to individual PDA plates.

Confluent PDA plates of all transformants were washed with 8 ml of 0.01% TWEEN® 20 and inoculated separately into 1 ml of MDU2BP medium in sterile 24 well tissue culture plates and incubated at 34° C. Five days after incubation, a 10 μl sample from harvested broth of each culture was analyzed using 8-16% Tris-glycine SDS-PAGE gels according to the manufacturer's instructions. SDS-PAGE profiles of the cultures showed that several transformants had a new major band of approximately 100 kDa.

A confluent PDA plate of one transformant for each transformation was washed with 8 ml of 0.01% TWEEN® 20 and inoculated into 500 ml glass shake flasks containing 100 ml of MDU2BP medium and incubated at 34° C. with agitation at 200 rpm to generate broth for characterization of the variants. The flasks were harvested on day 5 and filtered using a 0.22 μm GP Express plus Membrane.

Example 9: Purification of the Aspergillus fumigatus Family GH3A Beta-Glucosidase Variant L89M+G91L+F100D+I140V+I186V+S283G+N456E+F512Y and Variant L89M+G91L+I140V+I186V

Broth supernatant containing either A. fumigatus GH3A beta-glucosidase variant L89M+G91L+F100D+I140V+I186V+S283G+N456E+F512Y, or A. fumigatus GH3A beta-glucosidase variant G142S+Q183R+H266Q+D703G, obtained according to Example 8, was concentrated and desalted using ECONOPAC® 10 DG columns equilibrated with 20 mM Tris-HCl pH 8.0. A 3 ml volume of broth supernatant was loaded onto the column and then eluted with a 4 ml volume of 20 mM Tris-HCl pH 8.0. The desalted sample was purified using a 20 ml MONO Q™ column (HR 16/10, GE Healthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris-HCl pH 8.0. Desalted material was loaded onto the MONO Q™ column and then washed with 5 column volumes of 20 mM Tris-HCl pH 8.0. The A. fumigatus GH3A beta-glucosidase variant was eluted using a 20 column volume gradient from 0.0 to 0.40 M NaCl in 20 mM Tris-HCl pH 8.0. Fractions of 6 ml were collected, and fractions 35 through 50 were pooled for the variant A. fumigatus GH3A beta-glucosidase variant L89M+G91L+F100D+I140V+I186V+S283G+N456E+F512Y and fractions 39 through 59 were pooled for the A. fumigatus GH3A beta-glucosidase variant G142S+Q183R+H266Q+D703G. Purified samples were submitted to SDS-PAGE analysis using a 8-16% stain free Tris-HCl gel with imaging using a CRITERION® Stain Free Imager. The SDS-Page analysis showed the samples to be greater than 95% pure.

Example 10: Determination of the Specific Activity of the Wild-Type Aspergillus Fumigatus Family GH3A Beta-Glucosidase, Variant L89M+G91L+F100D+I140V+I186V+S283G+N456E+F512Y, Variant L89M+G91L+I140V+I186V, and Variant F100D+S283G+N456E+F512Y

Purified wild-type Aspergillus fumigatus Family GH3A beta-glucosidase and variant F100D+S283G+N456E+F512Y, purified according to Example 4, and variant L89M+G91L+F100D+I140V+I186V+S283G+N456E+F512Y and variant L89M+G91L+I140V+I186V, purified according to Example 9, were evaluated for their ability to enhance the hydrolysis of PCS by the “Trichoderma reesei cellulolytic protein composition” (Example 6).

The hydrolysis of PCS and the analysis thereof was performed according to the procedure described in Example 6.

The results shown in FIG. 2 demonstrated that the variants of the A. fumigatus Family GH3A beta-glucosidase have a higher specific activity than the wild-type beta-glucosidase (releases more glucose at a given enzyme concentration than the wild-type enzyme at the same concentration under the specified conditions).

The present invention is further described by the following numbered paragraphs:

[1] A beta-glucosidase variant, comprising a substitution at one or more (e.g., several) positions corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 2, wherein the variant has beta-glucosidase activity.

[2] The variant of paragraph 1, which is a variant of a parent beta-glucosidase selected from the group consisting of: (a) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii); (c) a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof; and (d) a fragment of the mature polypeptide of SEQ ID NO: 2, which has beta-glucosidase activity.

[3] The variant of paragraph 2, wherein the parent beta-glucosidase has at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2.

[4] The variant of paragraph 2, wherein the parent beta-glucosidase is encoded by a polynucleotide that hybridizes under high or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof or (ii) the full-length complement of (i).

[5] The variant of paragraph 2, wherein the parent beta-glucosidase is encoded by a polynucleotide having at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, or the cDNA sequence thereof.

[6] The variant of paragraph 2, wherein the parent beta-glucosidase comprises or consists of the mature polypeptide of SEQ ID NO: 2.

[7] The variant of paragraph 2, wherein the parent beta-glucosidase is a fragment of the mature polypeptide of SEQ ID NO: 2, wherein the fragment has beta-glucosidase activity.

[8] The variant of paragraph 2, which has at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, sequence identity to the amino acid sequence of the parent beta-glucosidase.

[9] The variant of any of paragraphs 1-8, which has at least 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 2.

[10] The variant of any of paragraphs 1-9, wherein the variant consists of 720 to 863 amino acids, e.g., 720 to 739, 740 to 759, 760 to 779, 780 to 799, 800 to 819, 820 to 839, and 840 to 863 amino acids.

[11] The variant of any of paragraphs 1-10, wherein the number of substitutions is 1-8, such as 1, 2, 3, 4, 5, 6, 7, or 8 substitutions.

[12] The variant of any of paragraphs 1-11, which comprises a substitution at a position corresponding to position 89.

[13] The variant of paragraph 12, wherein the substitution is Met.

[14] The variant of any of paragraphs 1-13, which comprises a substitution at a position corresponding to position 91.

[15] The variant of paragraph 14, wherein the substitution is Leu.

[16] The variant of any of paragraphs 1-15, which comprises a substitution at a position corresponding to position 100.

[17] The variant of paragraph 16, wherein the substitution is Asp.

[18] The variant of any of paragraphs 1-17, which comprises a substitution at a position corresponding to position 140.

[19] The variant of paragraph 18, wherein the substitution is Val.

[20] The variant of any of paragraphs 1-19, which comprises a substitution at a position corresponding to position 186.

[21] The variant of paragraph 20, wherein the substitution is Val.

[22] The variant of any of paragraphs 1-21, which comprises a substitution at a position corresponding to position 283.

[23] The variant of paragraph 22, wherein the substitution is Gly.

[24] The variant of any of paragraphs 1-23, which comprises a substitution at a position corresponding to position 456.

[25] The variant of paragraph 24, wherein the substitution is Glu.

[26] The variant of any of paragraphs 1-25, which comprises a substitution at a position corresponding to position 512.

[27] The variant of paragraph 26, wherein the substitution is Tyr.

[28] The variant of any of paragraphs 1-27, which comprises a substitution at two positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512.

[29] The variant of any of paragraphs 1-27, which comprises a substitution at three positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512.

[30] The variant of any of paragraphs 1-27, which comprises a substitution at four positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512.

[31] The variant of any of paragraphs 1-27, which comprises a substitution at five positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512.

[32] The variant of any of paragraphs 1-27, which comprises a substitution at six positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512.

[33] The variant of any of paragraphs 1-27, which comprises a substitution at seven positions corresponding to any of positions 89, 91, 100, 140, 186, 283, 456, and 512.

[34] The variant of any of paragraphs 1-27, which comprises a substitution at each position corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512.

[35] The variant of any of paragraphs 1-34, which comprises one or more substitutions selected from the group consisting of L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y, as described herein.

[36] The variant of any of paragraphs 1-34, which comprises or consists of the substitutions L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y.

[37] A composition comprising the variant of any of paragraphs 1-36.

[38] An isolated polynucleotide encoding the variant of any of paragraphs 1-36.

[39] A nucleic acid construct comprising the polynucleotide of paragraph 37.

[40] An expression vector comprising the polynucleotide of paragraph 37.

[41] A host cell comprising the polynucleotide of paragraph 37.

[42] A method of producing a variant, comprising: (a) cultivating the host cell of paragraph 41 under conditions suitable for the expression of the variant; and (b) recovering the variant.

[43] A transgenic plant, plant part or plant cell transformed with the polynucleotide of paragraph 38.

[44] A method of producing the variant of any of paragraphs 1-36, comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the variant under conditions conducive for production of the variant; and (b) recovering the variant.

[45] A method for obtaining a variant, comprising introducing into a parent beta-glucosidase a substitution at one or more positions corresponding to positions 89, 91, 100, 140, 186, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 2, wherein the variant has beta-glucosidase activity; and recovering the variant.

[46] A method for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of the variant of any of paragraphs 1-36.

[47] The method of paragraph 46, wherein the cellulosic material is pretreated.

[48] The method of paragraph 46 or 47, further comprising recovering the degraded cellulosic material.

[49] The method of any of paragraphs 46-48, wherein the enzyme composition comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[50] The method of paragraph 49, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[51] The method of paragraph 49, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.

[52] The method of any of paragraphs 46-51, wherein the degraded cellulosic material is a sugar.

[53] The method of paragraph 52, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.

[54] A method for producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition in the presence of the variant of any of paragraphs 1-36; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[55] The method of paragraph 54, wherein the cellulosic material is pretreated.

[56] The method of paragraph 54 or 55, wherein the enzyme composition comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[57] The method of paragraph 56, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[58] The method of paragraph 56, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.

[59] The method of any of paragraphs 54-58, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.

[60] The method of any of paragraphs 54-59, wherein the fermentation product is an alcohol, an organic acid, a ketone, an amino acid, or a gas.

[61] A method of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of the variant of any of paragraphs 1-36.

[62] The method of paragraph 61, wherein the cellulosic material is pretreated before saccharification.

[63] The method of paragraph 61 or 62, wherein the enzyme composition comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[64] The method of paragraph 63, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[65] The method of paragraph 63, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.

[66] The method of any of paragraphs 61-65, wherein the fermenting of the cellulosic material produces a fermentation product.

[67] The method of paragraph 66, further comprising recovering the fermentation product from the fermentation.

[68] The method of any of paragraphs 66 or 67, wherein the fermentation product is an alcohol, an organic acid, a ketone, an amino acid, or a gas.

[69] A whole broth formulation or cell culture composition comprising the variant of any of paragraphs 1-36.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. 

What is claimed is:
 1. A beta-glucosidase variant, comprising a substitution to a parent beta-glucosidase at positions corresponding to positions 100, 283, 456, and 512 of SEQ ID NO: 2, wherein the variant has beta-glucosidase activity, and wherein the variant comprises an amino acid sequence having at least 95% but less than 100% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 2. The variant of claim 1, wherein the substitution at a position corresponding to position 100 is with Asp.
 3. The variant of claim 1, wherein the substitution at a position corresponding to position 283 is with Gly.
 4. The variant of claim 1, wherein the substitution at a position corresponding to position 456 is with Glu.
 5. The variant of claim 1, wherein the substitution at a position corresponding to position 512 is with Tyr.
 6. The variant of claim 1, which comprises an Asp at a position corresponding to position 100 of SEQ ID NO: 2, a Gly at a position corresponding to position 283 of SEQ ID NO: 2, a Glu at a position corresponding to position 456 of SEQ ID NO: 2, and a Tyr at a position corresponding to position 512 of SEQ ID NO:
 2. 7. The variant of claim 1, which comprises an Asp at position 100 of SEQ ID NO: 2, a Gly at position 283 of SEQ ID NO: 2, a Glu at position 456 of SEQ ID NO: 2, and a Tyr at position 512 of SEQ ID NO:
 2. 8. The variant of claim 1, which comprises the substitutions F100D, S283G, N456E, and F512Y.
 9. The variant of claim 1, wherein the parent beta-glucosidase comprises an amino acid sequence having at least 95% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 10. The variant of claim 1, wherein the parent beta-glucosidase comprises an amino acid sequence having at least 96% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 11. The variant of claim 1, wherein the parent beta-glucosidase comprises an amino acid sequence having at least 97% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 12. The variant of claim 1, wherein the parent beta-glucosidase comprises an amino acid sequence having at least 98% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 13. The variant of claim 1, wherein the parent beta-glucosidase comprises an amino acid sequence having at least 99% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 14. The variant of claim 1, wherein the parent beta-glucosidase comprises amino acids 20 to 863 of SEQ ID NO:
 2. 15. The variant of claim 1, which comprises an amino acid sequence having at least 96% but less than 100% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 16. The variant of claim 1, which comprises an amino acid sequence having at least 97% but less than 100% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 17. The variant of claim 1, which comprises an amino acid sequence having at least 98% but less than 100% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 18. The variant of claim 1, which comprises an amino acid sequence having at least 99% but less than 100% sequence identity to amino acids 20 to 863 of SEQ ID NO:
 2. 19. An isolated polynucleotide encoding the variant of claim
 1. 20. A host cell comprising an expression vector, wherein the expression vector comprises the polynucleotide of claim
 19. 21. A method of producing a variant, said method comprising: (a) cultivating the host cell of claim 20 under conditions suitable for the expression of the variant; and (b) recovering the variant.
 22. A method for degrading a cellulosic material, said method comprising: treating the cellulosic material with an enzyme composition, wherein the composition comprises the variant of claim
 1. 23. The method of claim 22, said method further comprising recovering the degraded cellulosic material.
 24. The method of claim 22, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
 25. A method for producing a fermentation product, said method comprising: (a) saccharifying a cellulosic material with an enzyme composition, wherein the composition comprises the variant of claim 1; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
 26. The method of claim 25, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
 27. A whole broth formulation or cell culture composition comprising the variant of claim
 1. 